Initial researches on neuro-functional status and evolution in chronic ethanol consumers with recent traumatic spinal cord injury

We found differences related to the neuro-functional deficiency and clinical progress, among non-consumers and chronic consumers of ethanol, with recent traumatic spinal cord injury (SCI). We present a synthesis of related data on lesion mechanisms in post-traumatic myelogenous disorders, namely some of the alcohols and their actions on the nervous system, with details on the influences exerted, in such afflictions, by the chronic consumption of ethanol. The subject is not frequently approached – according to a literature review with systematic elements, which we have done before – thus constituting a niche that deserves to be further explored. The applicative component of the article highlights statistical data resulted from a retrospective study regarding the specialized casuistry from the Neuromuscular Recovery Clinic of the “Bagdasar Arseni” Emergency Clinical Hospital, following the comparative analysis of two groups of patients with recent SCI: non-consumers – the control group (n=780) – and chronic ethanol consumers – the study group (n=225) – with the addition of a prospective pilot component. Data processing has been achieved with SPSS 24. The American Spinal Injury Association Impairment Scale (AIS) mean motor scores differ significantly (tests: Mann-Whitney and t) between the control and study group in favor of the second, both at admission (p<0.001) and at discharge (p<0.001). AIS mean sensitive scores differ between the two lots, and also in favor of the study, but statistically significant only at discharge (p=0.048); the difference at admission is not significant (p=0.51) – possibly because of alcoholic-nutritional polyneuropathy. These findings, with numerous related details, later presented in the text, are surprising, which requires further studies and attempts of understanding.


Introduction
Data on lesion mechanisms in post-traumatic myelin conditions are presented in various papers in the literatureincluding older ones, written by one of the authors (and contributors) of this article [1] -with details and some new elements, as the research progresses in this area (some minimal general references to this subject are also found in a recent article [2] of the first author of this article). From this perspective, in the introductory part of this journalistic approach, we (re-)emphasize, very concisely, a few important current elements of (morpho-)physiopathology in traumatic spinal cord injury (SCI).
Following spinal cord injury, changes occurred almost instantaneously, constituting primary lesions, consecutive, of the nervous tissue: mechanical aggression (compression/traction/torsion) at the cellular level, axonal ruptures -with retractions, dynamically and immunomolecularly induced ("retraction bulbs" [3] / "retraction clubs at the ends of severed axons" [4]) and expulsion of tissue fragments [5] -but also deterioration of blood vessels from microcirculation of the gray matter [6] with iron release [which contributes to secondary lesions by enhancing the production of toxic free radicals/reactive oxygen species (ROS): Fenton reactions [7,8] -by the hemorrhages thus produced -including central (hemorrhagic) necrosis [9], central myelin dislocation at the site of the injury [9] including in the region of the ependymal duct -membranal "impact" [10] depolarization of neurons and gliocytes (by abundant release of potassium ions from lacerated cells and inadequate glutamate release: intense excitotoxicityinducing processes) and local accumulation of dynorphin (with opioid receptor activation -also generating secondary lesions: especially ischemia [11]) as well as glutamic acid [12].
It is a known fact -and we already have begun their succinct presentation in this synthetic paradigm -following an injury at the level of the central nervous system (CNS), including at the myelin level, primary and secondary lesions develop [13]. The transition to "the cascade" [14] of secondary lesion events comprisesin summary -the calpain -dependent activation [14,15], of the NMDA (N-methyl-D-aspartic acid) and AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid) [11] receptors with the consecutive intracytoplasmic accumulation of calcium ions. These, together with the induction, by means of aggressive reaction mechanisms from the category listed above, of an excessive, and therefore unsustainable, a local metabolic augmentation, as a consequence -as important as detrimental -the production, at the mitochondrial level, in marked quantities, of ROS [16,17], the "oxidative stress" being a real "hallmark" [18] of the redoubtable (secondary) lesional mechanisms of SCI -and not only -which can lead, finally, to insurmountable cellular energetical depletion with the subsequent triggering of the apoptosis mechanisms [19]. The picture of the secondary morpho-/physio-pathological events is amplified including with some vicious cycle developments by triggering of the inflammatory reactions (with consecutive local acidosis), in the complex context of change, in a negative sense, of the local micro ambient: the main cause of the tissular endogenous regenerative trends failure, however, very small and/or improper from the CNS level, in particular of the spinal cord [20]. Damage in the blood vessels and myelin (in the injured area) augments the picture of secondary lesions, by local ischemia, intraneuraxial extravasation of macrophages derived from monocytes with their infiltration into the lesioned myelitic tissue [21] and the occurrence of demyelination/axonal degeneration [11]. At the same time, at the local-regional level (additional "guarantees" of the desolate irreversibility of the whole lesional phenomenon -as the neurons do not reproduce [22]), cellular necrosis occurs immediately, by the destructive mechanical effect of trauma, and by osmolysis subsequent to post-compressive and vasogenic/ ischemic edema [23][24][25] processes of apoptosis, "apoptosis-like" (the latter especially by "protein misfolding" [7,14]) with the subsequent diminution of the number of neuronal cellular units -interruption or disconnection of intramedullary pathways of communication (extensions of affected perikaryons) and fibrous scarring/gliosis ("reactive astrogliosis" [26], "fibroglial scar" [23]) -all followed by neuro-functional deficit: motor and/or of sensitivitysomatic and/or vegetative (in different degrees) [11] -at sub-lesional level.
Taking into account the individual [27] and social [28,29] implications of the consequences of the traumatic spinal cord injuries (SCI), various therapeutical modalities were/are under study -of which, in their majority, one of the authors (and contributors) of the current article has [30] already written and published in detail intended to positively influence the clinical (acute/sub-acute/chronic/sub-chronic) progression of these patients -but all, till now, without spectacular curative effects [31].
In parallel, another type of molecule is being researched, potentially having beneficial effects in the treatment, and subsequent post-acute/sub-acute recovery of SCI -polyethylene glycol (PEG). PEG applied to the area of myelogenous injury in adult Guinea pigs produced very good neuro-functional results by having a protective (sealing) effect against damage to the spinal neuron axolemmas of the tested animals [32,33]. Other studies on Guinea pigs were conducted and showed the benefits in the evolution of lesions after SCI when a PEG single dose subcutaneous injection was administered [34]. PEG would not act as a neutralizer of excitotoxic products in medullary lesions [33], but it would reduce perilesional excitotoxicity by mitochondrial membrane stabilization (after its intra cytosolic entry into the neurons situated in the vicinity of the injured neurons) [33,35]. PEG applied to extraneuraxial lesioned axons (of the peripheral nerves) would act beneficially for restoring the normal nervous tract by rapidly restoring (after post-lesional PEG application) axonal continuity [34,36]. The PEG solution works by removing water from cell membranes, which allows them to merge [37].
The use of PEG or a related copolymer with a polymerization index (188) and a molecular weight of 3500 Daltons has been investigated -also in the treatment of several complete myelogenous lesions in larger animals, such as dogs, -and it was found that: "… polymer injection is a safe adjunct to the conventional management of severe neurological injury in dogs. We did not observe any unacceptable clinical response to polymer injection ..." Outcome measures over the 6 to 8-week trial were improved by polymer injection when compared to historical cases [38].
The patients with chronic alcoholism display -as an observation factor resulting from the experience of our casuistry (from the Neuromuscular Recovery Clinic of the lesions of the intraneuraxial white matter are produced (without being correlated with significant structural changes in the lipids of myelin composition) [48]. The entire histopathological and functional encephalic picture resembles a process of accelerated neural aging [48].
It is also known that peripheral nervous system lesions -chronic alcoholism also being able to produce symmetrical sensory and motor polyneuropathiescan favorably affect the axonal regeneration in the deteriorated central nervous system by IL6 participation, with the activation of the transcription factors cREB (cAMP response element-binding protein) and ATF3 (Cyclic AMP-dependent transcription factor) [1,49]. Research has also been conducted on the effect of neurotrophin 3 (NT3) in the case of post-traumatic myeloradicular disorders. NT3 is a neurotrophic protein that can improve motoneuronal synaptogenesis which would perhaps explain the discrepancy between the objectified results of the post VMT: much less severe, between the motor scores present at the chronic alcoholics towards the nonchronic consumers of ethanol -distal to a myelo-contusive zone [50,51]. The thorough research on the implications of alcohol abuse shows that NT3 has higher values in males who are chronic alcohol consumers in comparison with patients without the vice of drinking [52].
However, the effects of ethanol are not fully elucidated, and a dynamic connection of glutamate with ethanol intake is also observed [53]. The time required to pass from occasional alcohol consumption to alcohol abuse/ethanolic dependence depends on the individual mental and neurostructural character ("addiction is a voluntary operant behavior that results from a temporally myopic view of available alternatives in a choice situation") [53].
In the chronic phase of ethanol dependence, the nervous system decreases its responsiveness to the most important neuro-excitant: glutamate, along with modifications in the conformation of the gammaaminobutyric acid (GABA) receptors and of the N-methyl-D-aspartate (NMDA) receptors [53]. Due to the decrease in the representation of NMDA receptors in the neural membrane, chronic alcohol consumption causes the excessive production of glutamate in a compensatory way (being a factor that contributes to the morpho-functional changes in ethanol dependence syndrome) [53].
The alcohol molecules are made up of carbon, hydrogen and oxygen atoms bonded through sigma covalent bonds, with the formation of intra-/intermolecular hydrogen electrostatic bonds, where the components are of different mass ratios [54][55][56]. From the molecular point of view, the ethanol and polyethylene glycol structures are related, (both being part of homologous series of the functional class of the organic substances called alcohols) Bagdasar Arseni Clinical Hospital) -a progression and a more favorable prognosis of myelin lesions compared to similar non-ethanol consumer patients (see below).
Chronic alcoholism is defined as a long-term exposure (greater than 14-35 days in the case of laboratory animals -rats) [39,40] at high doses of ethanol. In the case of people, excessive ethanol doses are defined as: "... 8 or more drinks per week for women and 5 or more drinks on any day or 15 or more drinks per week for men ..." [41]), respectively, of more than 8 standard daily ethanolic units (standard drink per day unit = 10 g ethanol) [42]. Taking into account the lack of unitary data on the definition of chronic alcoholism, in the retrospective component we used the diagnostic verbalizations of the doctors (even our own interview experience revealed the positive answers of the interviewed people by their state regarding ethanol consumption as occurring for years). Following the analyzation of literature data, including those on fundamental research elements (specific laboratory techniques related to the approached subject), we extrapolated the calculations regarding the duration of the occurrence of chronic alcoholism in humans at an average duration of about 2.5 years (considering that the equivalence of the life span between man and rat is of 38.4 human days for a rat's day of life) [43].
Ethyl alcohol affects the development of the human body at all stages, starting with intrauterine life, influencing individuals of all ages, sex, lifestyle or other comorbidities. Numerous studies have been conducted on the negative effects of ethanol abuse on human growth and development, on discernment, on the destructive pathophysiological consequences (at the level of the entire body). During pregnancy, the blood ethanol concentration, at the fetus level, is approximately two third of the mother's blood [44]. Maternal consumption of ethanol during gestation predisposes the fetus to the risk of developing fetal alcohol syndrome, which may include: diminishing fetal weight, ataxia, intellectual retardation and somatic growth, facial dysmorphism, behavioral disorders [45][46][47]. Regarding the development of the nervous system in the intrauterine life, the mechanisms by which substances abusively consumed (including ethanol) negatively influence this phenomenon are not clarified, but alcohol is considered to affect normal neurotransmitter-receptor interaction, followed by functional consequences of the neurotransmitters' actions in adult life [46].
A physiopathological neurotoxic ethanolic mechanism can also be encountered in the case of Wernicke encephalopathy (consequence of the thiamin deficiency resulting from malnutrition that occurs in chronic ethanol abuse) [48]. In this case, in addition to the psycho-cognitive disturbances (included in the Korsakoff syndrome), database (not ISI indexed) and 23 in the PMC of which 16 ISI indexed (Table 4 of Annex I).
Thus, we have learnt that the target subject of our research is not widely found in the literature. In a brief summary of the tangent problems, there are opinions that the post-VMT recovery process in ethanolic patients is more difficult than in non-alcoholic patients -but not due to reasons linked to the intrinsic neuro-functional reactivity of the spinal cord towards traumatic lesions, but for predominantly contextual reasons, related to the lower cooperation of such patients in the psycho-behavioral aspect, as well as their increased risk of falling, with possible subsequent physical trauma, including spinal cord-injury, in the context of abundant inebriation episodes, with balance disturbances (to which chronic ethanol impregnation of the nervous system can also bring its contribution [47]). In addition, there are studies regarding a possible propensity, for chronic alcoholics with SCI, for neuropathic pain (the list of articles selected by means of the above method can be found in alphabetical order in the appendix).
In order to objectify the connection between the chronic consumption of ethanol and the (acute/sub-acute) progression of patients with spinal cord injury, we performed a retrospective study of patients admitted in the Neuromuscular Recovery Clinic with SCI diagnosis during the period between January 2005 and August 2018 -a total of 1005 patients of which 196 women, 809 men aged 18-95 years (Figures 1 and 2 of Annex II). To quantitatively objectify the post-SCI neuro-functional deficits for each of the patients analyzed, we applied the AIS standardized assessment scale and the associated (synthetic) Frankel grading in each of the patients analyzed. The AIS (American Spinal Cord Association Impairment Scale) scale quantifies (through scores) the post-SCI motor and sensitivity deficits; while the Frankel scale is very useful for assessing the patient's functional disorders (using gradations from A to E) [74][75][76].
For the statistical processing of primary data required for this study, we used the "Statistical Package for Social Sciences" SPSS 24.0 (sequentially accessed in 2018 and 2019) and Microsoft Excel 2010.
In addition to the descriptive statistics elements, there were two stages in the data processing. First, the patients were separated into two groups -ethanol chronic consumers and non-chronic ethanol consumers which are sub-divided into sub-groups, depending on gender, age, environment, causes for the occurrence of SCI, pathophysiological mechanisms of S-C traumatic lesions, myelogenous lesion level, neurological/neuro-dysfunctional level. In the second stage, we performed a comparison through differentiation tests -the patients' status (chronic ethanol consumers with several common physio-biochemical properties like hydrosolubility, ability to cross the hemato-neuraxial barrier which is explained in Tables 1-3 of Annex I. From the physio-pathological point of view, in the recovery after SCI, the astrocytes have a very important role, in (in the acute post-traumatic phase), promoting the formation of perilesional glial scars (starting with the sub-acute phase) [57]. PEG administered after post-SCI favors the functional recovery of the nerves, through the protection of neuronal growth cones and being hindered in its action by neuronal astrocytic perilesional scars [57]. As it is known that alcoholism disturbs the function of astrocytes, a neuroprotective effect of ethanol could be explained in vertebral medullary traumatology [58].
At the same time, a neuroprotective effect of ethanol consumption has also been observed, by inhibiting the formation of harmful ceramide membranes [59]. Ethanol diffuses easily through the neuronal membranes, bonding with both sub-membrane lipid domains and intracellular organisms [60]. In a similar way, it seems that PEG molecules interact with neuronal membranes, providing a neuroprotective effect in the experimental SCI models [61].
Hence, our research endeavor based on clinical and statistical evidence attempts to make further progress by means of applied laboratory tests in our future studies in providing possible explanations of the similarities between these two substances, in terms of their beneficial effects in the case of sub-acute lesions resulted from SCI in chronic ethanol consumers (Tables 1-3 of Annex I).

Materials, Methods and Results
In We used the Kolmogorov-Smirnov test to verify, from a statistical point of view, the acceptability (or not) of the normality of the data distribution (Tables 16 and 17 in Annex I show the results of the tests performed for the motor AIS score values on admission). From these tables we only take the "final" information, namely the degree of statistical significance: p=0.024 and p<0.001, respectively. In this kind of test, the confirmation of data normality is only accepted if the grade is "high" (over a set threshold in SPSS of 0.20), which is not the case. The unacceptability of data normality does not validate the use of statistical parametric type tests (such as test t). However, the use of non-parametric tests (based on ranks, medians) such as the Mann-Whitney test, remains possible. An example of its application is presented in Tables 18 and 19 (Annex I).
From Table 18 (Annex I) we get information about the highest mean rank which will indicate the "privileged" group, in our case the group of chronic ethanol consumers, and from Table 19 (Annex I) we can only take the degree of statistical significance: p<0.001. In this kind of tests, the confirmation of the statistically significant difference and non-chronic ethanol consumers) and we subsequently performed a comparison of their progress, using the tests: Kolmogogoro-Smirnov, Mann-Whitney/Wilcoxon, chi-square, Fisher exact and Z. The values obtained were statistically significant for different cases where p<0.05. For the specific calculations, confidence intervals (CI) associated with a trustworthiness level of 95% were considered [77,78].
Given the editorial space, naturally limited, we will present in this article all the ample/exhaustive volume of primary data and consecutive results obtained. The data that we consider to be the most important is the essential data referring to the issues addressed in our study (aspects related to the comparison between the two groups mentioned, the quantified assessments of the neurofunctional deficits and the assessment of the patients).
Knowing that AIS motor and sensory scores analytically measure the post-lesional myelogenous neuro-functional deficit, and that the associated Frankel scale grading classifies, synthetically, from A to E, the neuro-functional status, we grouped post-SCI patientswhether or not they were chronic ethanol consumers -a patients with on SMAST 3 score to that of non-alcoholic patients, we get a percentage of 81.25% ( Figure 6, Annex II). Thus, we find percentage values which are very close to those identified by us in the retrospective study; moreover, the statistical analysis for differentiation shows that the distribution between chronic ethanol consumers and non-chronic consumers (identified using the SMAST quantification) found in the retrospective analysis of the data from the medical records does not differ significantly statistically. This results from the application of the chisquare goodness of fit test: the p-value obtained is 0.829 if the test scores of 3 are not taken into account, and it becomes 0.804, if the patients surveyed are predominantly considered non-chronic ethanol consumers (3 of the 4 patients who got a 3 score on the SMAST testing). These do not contradict the narrative collection for the necessity of the primary data from the retrospective study.

Conclusions
Altought quite paradoxaly, overall our findings are, we consider, very intersting and worth suplementary research.

Conflict of Interest
The authors confirm that there are no conflicts of interest. between the groups is only accepted if this degree is "low" (below the conventional threshold of 0.05). Therefore, the means of the American Spinal Injury Association Impairment Scale (AIS) motor scores differ significantly (Mann-Whitney and t) between the control groups and study groups on admission (p˂0.001). The same conclusion is valid for discharge data.
Regarding AIS mean sensitive scores, they differ between the two lots, also in favor of the study lot, but statistically significant only at discharge (p=0.048); the difference at admission is not significant (p=0.519) possibly because of alcoholic-nutritional polyneuropathy "… usually prominent sensory features" [79] (Annex I, Table 20).
Regarding the patients' progress belonging to the control and study groups -between admission and discharge reflected by the AIS motor scores, respectively AIS sensitivity, there is a statistically significant difference, in favor of the study group (p<0.001, Table 21 in Annex 1). More specifically, the control group has a mean motor score increase of just 1.50 points and the study group has a mean motor score increase of 2.99 points (bigger); regarding the sensitive score, for the control group there is mean increase of 5.76 points less than that for the study group which is a mean increase of 7.69 points. (Annex 1, Table 21).

Discussion
The fact that the patients in the analyzed groups did not have a standardized diagnosis of chronic alcoholism is a relative limitation of this study. After analyzing retrospectively the related clinical medical records, one could not find any such classifications there. That is why we have used, for this purpose, representative definitions from the literature. However, for the sake of rigor we will carry this out following the present research endeavor, in a prospective future extension which carried out on a small pilot sample, the assessment of a number of 16 patients with recent SCI, the discrimination between the state of chronic alcohol consumer and non-chronic consumer, by means of the test used internationally for the assessment of ethanol consumption: Short Michigan Alcohol Screening Test (SMAST) [80,81]. Thus, we tested patients admitted at the time (on 1 March 2019) in our clinic for recovery after acute/sub-acute spinal cord injury. Specifically, we interviewed 2 women and 14 men. According to the quantification by means of the SMAST questionnaire, the score value 3 is not interpretable as chronic alcoholism, but it is considered "borderline". Nine patients (2 females and 7 men) got the 1 or 2 score, 4 patients (all men) got the 3 score and the others got a score ≥4; adding the number of Annex I                       Annex II